The overall goal of this proposal is to develop an imaging agent and detection system for labeling and real- time detection of residual cancer in the tumor bed during sarcoma resection surgery that will be quickly adopted by surgeons/hospitals because it can eliminate or significantly reduce secondary surgeries and local recurrence. Failure to remove cancer cells during surgery is a leading risk factor for local tumor recurrence and subsequent reoperation. For 35% of sarcoma patients, limb-sparing surgery supported by frozen sections, which only analyze a small fraction of the resected tumor, results in local recurrence in the absence of adjuvant therapy, indicating microscopic residual cancer has been left behind. Furthermore, in other cancer surgeries, as in breast lumpectomies, positive margins and secondary surgeries can occur more than 50% of the time. Technology that can assess whether tumor cells have been adequately removed during surgery can have major impact on cancer therapy worldwide as more than two million people undergo cancer surgery every year. Such technology can reduce rates of local recurrence and eliminate the need for secondary surgeries and adjuvant radiation, reducing associated healthcare costs. Lumicell has developed an injectable imaging agent and hand-held imager that can detect single cells with a wide field of view. Our novel detection technology, developed at MIT and licensed to Lumicell, reliably detects microscopic residual cancer cells in vivo during sarcoma resection surgeries in mice. Based upon mouse and dog tests and using our computer simulation of the imaging agent performance we forecast a peak contrast in humans at 36-48 hours from injection. This timeframe is not ideal with the current preoperative procedures for cancer surgery and we wish to re-engineer the imaging agent to achieve the maximum tumor-to-background signal contrast within 18-24 hours from injection. We propose to re-design our cancer-activatable imaging agent with enhanced pharmacokinetics to rapidly reach the tumor and to increase clearance rates from the blood. To reach the level of signal that we believe can be achieved, we propose on Specific Aim 1 to engineer several designs of imaging agents and to characterize their performance in mice and dogs, and then simulate their performance in humans. On Specific Aim 2 we will select one or two candidate imaging agents with the desired performance between 18-24 hours after injection and conduct validation studies in mice (surgery and pathology). PUBLIC HEALTH RELEVANCE: The proposed project aims to address the unmet clinical need for intraoperative assessment of residual cancer cells in the tumor bed after gross tumor resection in cancer patients. A novel method for fast and thorough examination of the tumor bed in real-time will be developed by engineering an imaging agent suitable to for the typical hospital surgical protocols. This will require the refinement of the Lumicell molecular imaging agent design to achieve peak activation in 18-24 hours (current performance is 36-48 hours) from injection. This technology has the potential to save lives, prevent secondary surgeries, minimize patient discomfort and surgical risks, and provide substantial savings in healthcare costs.